The present disclosure relates generally to microfluidic technology. More particularly, the present disclosure relates to self-loading microfluidic devices. The present disclosure further relates to methods of using the self-loading microfluidic devices for chemical assays and biological assays.
The search for new therapeutic agents involves rapidly screening test compounds in chemical assays and biological assays to identify lead compounds for further development. Once lead compounds are identified, additional screening may be needed to identify therapeutically effective amounts and toxicity levels. Screening therapeutic agents is important for patient treatment and for preventing the prescription of ineffective pharmaceuticals. For example, determining the minimum inhibitory concentration (MIC) of antibiotics against bacteria is important for preventing the prescription of ineffective antibiotics that may lead to the spread of antibiotic-resistant strains of bacteria. The minimum inhibitory concentration (MIC) of a compound is generally defined as the lowest dose that inhibits the growth of a cell during a set time interval. Determining the MIC for microbes is particularly important because of the increase in antibiotic-resistant bacteria and the decline in the discovery rate of new antibiotics.
Determining therapeutically effective concentrations including, for example, the MIC of antibiotics against bacteria is generally performed using diffusion or dilution methods. In diffusion methods to determine the MIC of microbes, for example, a hydrophilic strip or disc is infused with antibiotic and placed in contact with the surface of an agar plate upon which a microbe is growing. The antibiotic diffuses radially through the agar gel and forms a concentration gradient that inhibits microbial growth close to the strip or disc. The formation of a visual ‘zone of inhibition’ in this assay enables the estimation of the MIC. Diffusion-based assays are technically simple to perform, however they have several disadvantages. For example, the results of the assays must be standardized to the specific characteristics of the agar growth media, which strongly influence diffusion of the antibiotics. Additionally, the analysis of these assays is subjective and variable.
Dilution methods to determine the MIC of microbes, for example, involves inoculating microbes into a series of separate culture tubes or onto separate agar plates containing nutrient media and a two-fold serial dilution of an antimicrobial agent. The Clinical Laboratory and Standards Institute (CLSI) in the United States and by similar institutions in other countries publish guidelines for dilution-based methods for determining MIC values. The MIC is determined by identifying the lowest concentration of antibiotic that inhibits microbial growth, and is typically measured by visual inspection. These assays are well characterized and provide a more quantitative readout than diffusion methods, however, they are generally more labor intensive. The use of a liquid handling robot to prepare dilution series in multiwell plates can reduce the time and labor involved, however these instruments are expensive and not widely available in laboratories.
Screening may also be used to conduct other chemical assays and biological assays such as, for example, analyzing cellular samples, performing and analyzing enzymatic reactions, identifying binding partners, and detecting target molecules. Immunoassays, for example, are commonly used for molecular recognition based on the specific interaction between an antibody and its antigen. Strip immunoassays have been successful for point-of-care clinical diagnosis because of its ease of use and rapid and sensitive format. However, the strip immunoassay may not be applicable for all immunoassays, and thus, many immunoassays are conducted using microtiter plates, which may require longer assay times and increased reagent use. Amplification of nucleotides, such as, for example, polymerase chain reaction (PCR) is commonly used, for example, to increase the copy number of nucleic acids to determine the nucleotide sequence of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, to alter and mutate particular bases, to determine whether a particular sequence is found in a cell or organism, to introduce restriction enzyme sites, and to quantitatively measure DNA and RNA molecules.
Microarrays are another format for analyzing nucleic acids and proteins. For example, a DNA array is a collection of spots having a DNA fragment at each spot. Arrays allow for a complementary strand of DNA or RNA possibly found in a sample to bind to the spotted DNA to detect binding. Microarrays may also be used for gene expression profiling in which isolated RNA is converted to a labeled cDNA that is hybridized to an array and detected. Arrays may also be used with proteins such as, for example, antibodies to detect antibody-antigen interactions.
Screening large numbers of samples presents a number of problems such as the high cost of equipment, high reagent requirements for performing the assays, and high volumes of solutions. Microfluidics technology represents an emerging area that permits the control and manipulation of fluids at a very small scale as well as the design of systems using small volumes of fluid. Thus, microfluidic technology addresses some of the drawbacks associated with the high cost of equipment, high reagent requirements for performing dilution-based and diffusion-based assays, and the high volumes of solutions needed.
The supply of samples and reagents to microfluidic devices commonly use pressure-driven and electrokinetic-driven pumping methods. These methods, however, require external power sources and other equipment, which can lead to added expense and limit point-of-care applications where the added equipment is unavailable or is of limited access. Power-free microfluidic pumping methods such as, for example, droplet-based passive pumping, evaporation, capillary flow, and gravity-driven flow, do not require external power sources, but may be limited by other requirements such as, for example, device priming, specific temperature and humidity, and surface treatments. Additionally, degas-driven flow is a phenomenon used to manipulate fluids in poly(dimethylsiloxane)-based microfluidic devices that does not require external power.
A microfluidic platform that uses low fluid volume consumption, better process control, and requires no additional external equipment for its operation may reduce costs, reduce user error, provide faster analysis, and provide a universal assay for use in a range of different environmental contexts. Accordingly, there exists a continued need to develop alternative microfluidic devices and methods of using microfluidic devices.